title: Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation.
abstract: Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.
authors.affiliation: Graduate Group in Biophysics, Physical Biosciences Division, Lawrence Berkeley National Laboratory and the Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Batchelor
authors.forename: Michaeleen
authors.initials: M
authors.lastname: Doucleff
authors.forename: Chul-Jin
authors.initials: CJ
authors.lastname: Lee
authors.forename: Koshi
authors.initials: K
authors.lastname: Matsubara
authors.forename: Sacha
authors.initials: S
authors.lastname: De Carlo
authors.forename: Johanna
authors.initials: J
authors.lastname: Heideker
authors.forename: Meindert H
authors.initials: MH
authors.lastname: Lamers
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Pelton
authors.forename: David E
authors.initials: DE
authors.lastname: Wemmer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D019295:Computational Biology; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012996:Solutions; D015534:Trans-Activators; D000255:Adenosine Triphosphate